Aire regulates central T cell tolerance by controlling thymic medullary epithelial cell (MEC) expression of a battery of transcripts encoding peripheral-tissue antigens. From early on, Aire was recognized to be a transcriptional regulator, but it soon became apparent that it does not operate like a conventional transcription factor, i.e. binding to promoters and inducing initiation of transcription. Studies completed during the last funding-cycle demonstrated that Aire-induced genes occur as intra- or inter-chromosomal clusters within individual cells, that Aire participates in multiple multi-protein complexes, and that it preferentially localizes to and activates so-called ?super-enhancers.? Super-enhancers (a.k.a ?stretch? or ?serial? enhancers) are extended regions of chromatin that are over-loaded with general and cell-type-specific transcription factors, and are thought to serve as depots for coordinate and efficient delivery of these factors to targeted promoters via chromatin looping. In addition, preliminary data documented herein indicate that Aire associates, directly or indirectly, with cohesin, one of the major orchestrators of long-range chromatin interactions (in collaboration with CTCF and/or mediator). Thus, the overall goal of this proposed project is to determine how Aire integrates into the three-dimensional organization of chromatin. This goal will be addressed via three Specific Aims: ? To determine whether Aire associates with proteins known to orchestrate 3D chromatin interactions: CTCF, cohesin, NIPBL and mediator. Experiments under this Aim will employ primarily biochemical approaches to determine whether Aire associates with the three major 3D-chromatin-organizing elements (and a cohesin-loader, NIPBL). ? To integrate Aire?s distribution along MEC chromatin with those of CTCF, cohesin and mediator; to determine how Aire impacts CTCF/cohesin/mediator placement and, vice versa, how cohesin influences Aire?s placement. This series of experiments will exploit recent improvements in ChIP-seq (and other whole-genome) technologies to map the binding sites of the three chromatin organizers in relation to Aire binding and other landmarks ? in wild-type, Aire-knockout and inducible Smc1-knockdown mice ? To define the relationship between Aire and three-dimensional chromatin organization. This set of experiments will use HiC technology to generate whole-genome interaction maps for MEC chromatin from Aire+/+ and Aire-/- mice. Successful completion of these studies will bring our understanding of Aire control of T cell tolerance to a new level of molecular understanding, and is likely to resolve several of the outstanding conundrums related to Aire function. The Aire story continues to intrigue!

Public Health Relevance

This study focuses on the molecular modus operandi of Aire, which is encoded by the gene mutated in individuals with APECED (or APS-1), a primary immunodeficiency disease characterized by multi-organ autoimmunity. While APECED patients are rather rare, the Aire pathway has been implicated in several of the more common autoimmune disorders -- including type-1 diabetes, myasthenia gravis, vitiligo and pernicious anemia -- reflecting polymorphisms either in Aire target-loci or in the gene encoding Aire itself. Manipulating new molecular interactions and pathways uncovered in this study may eventually have therapeutic potential; in the meantime, a more precise understanding of central tolerance mechanisms should result.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Mallia, Conrad M
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Harvard Medical School
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Bansal, Kushagra; Yoshida, Hideyuki; Benoist, Christophe et al. (2017) The transcriptional regulator Aire binds to and activates super-enhancers. Nat Immunol 18:263-273
Mathis, Diane; Benoist, Christophe (2016) Promiscuity Promotes Tolerance. J Immunol 196:2913-4
Yoshida, Hideyuki; Bansal, Kushagra; Schaefer, Uwe et al. (2015) Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells. Proc Natl Acad Sci U S A 112:E4448-57
Giraud, Matthieu; Jmari, Nada; Du, Lina et al. (2014) An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription. Proc Natl Acad Sci U S A 111:1491-6
Yang, Siyoung; Bansal, Kushagra; Lopes, Jared et al. (2013) Aire's plant homeodomain(PHD)-2 is critical for induction of immunological tolerance. Proc Natl Acad Sci U S A 110:1833-8
Giraud, Matthieu; Yoshida, Hideyuki; Abramson, Jakub et al. (2012) Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. Proc Natl Acad Sci U S A 109:535-40